The present invention relates to a novel pectic substance and a purification method thereof. More particularly, the present invention relates to pectic polysaccharides isolated from Angelica gigas Nakai, which are immuno-stimulative, and a purification method thereof. Also, the present invention is concerned with use of the pectic polysaccharides in the treatment or prophylaxis of immune-related disease and in basic immunology research.
The immuno-stimulating agents derived from natural substances are used for the treatment of cancers, AIDS and chronically infected states by enhancing immunoresponses or restoring lowered immune functions. Extensive research on fungi, bacidiomycota and medicinal herbs has been made to obtain immuno-stimulating agent. Particularly, the polymer fractions of the natural substances showed immuno-stimulation, such as anticancer activity, anticomplementary activity, and induction of lymphocyte proliferation. Glucan polysaccharides, such as lentinan, PSK, and schzophyllan, derived mainly from mushrooms, are useful to be clinical usefulness in therapies of cancer.
It is reported in oriental medical documents that Angelica, belonging to family Umbelliferae, can be used for the treatment of gynecological diseases (anaemia, interruption in blood circulation, etc) by virtue of anodynic effect. From Angelica gigas Nakai, found only in Korea, coumarin substances, such as decursin, decursinol, nodakenetin, umbelliferon, nodakenin, and xcex2-sitosterol, have been extracted, thus far.
Pharmaceutical research on the herb showed that the ether extract of Angelica gigas Nakai made the enucleated intestinal canals and uterus of rabbit excited. Free decursin and decursinol were reported to have a paralysis action on the enucleated intestinal canals of rabbit, a suppressive action on the enucleated heart of frog, and a breath-suppression and blood-depression action on rabbit. Recently, decursin have been found to have in vitro therapeutic activity against cancers and to enhance the activity of protein kinase C.
Bearing the above-mentioned situations in mind, the present inventors repeated thorough and intensive research with the aiming of finding a novel immuno-stimulating entity in Angelica gigas Nakai and finally, isolated a polysaccharide component which is immuno-stimulative and active against cancer.
Therefore, it is an object of the present invention to provide pectic polysaccharides which are immuno-stimulative and anticancer-effective.
It is another object of the present invention to provide a method for purifying the polysaccharides from Angelica gigas Nakai.
It is a further object of the present invention to provide information for use of the polysaccharides in the treatment or prophylaxis of cancers and immune-related diseases and in the recovery of immune functions.
It is still a further object of the present invention to provide information for use of angelan as an immuno-stimulating agent useful for basic immunology research.
In accordance with the present invention, the pectic polysaccharides which are purified by refluxing Angelica gigas Nakai in hot water and subjecting the hot water extract to ion exchange chromatography, are immuno-stimulative and shows anticancer activity.
A detailed description will be given of a purification method of angelan, a pectic polysaccharide, from Angelica gigas Nakai and the physicochemical characteristics thereof, below.
Sliced roots of Angelica gigas Nakai were refluxed with hot water for 1 hr, followed by filtering the water through a four-folded gauze and Whatman filter paper. The filtrate was mixed with three volumes of ethanol and allowed to stand at 4xc2x0 C. for 3 hours to give precipitates. They were obtained as brown polymers by centrifugation. From the fact that the polymers can easily obtained by ethanol precipitation and there are no denaturated protein precipitates even if solubilized precipitates in water are boiled for 20 min, the polymer fractions are assumed to contain a large amount of non-proteinous polymeric materials. Most of the colored material is absorbed in DEAE-cellulose, an anion exchange resin. Using the DEAE-cellulose absorption method, acidic and neutral fractions can be obtained. The acidic fraction was named angelan. They both are polysaccharides with a small amount of proteins. Angelan 10 KD in molecular weight was chemically characterized as a single substance as certified by the analytical high performance liquid chromatography method (HPLC).
Pectic polysaccharides compose most of the immuno-stimulating fraction of the hot water extract, amounting 85-90% of the total weight. The immuno-stimulating fraction also contains 7-8% of proteins and 15.5-68% of uronic acid. The fraction is also rich in calcium ion and magnesium ion in combination with other inorganic compounds including iron, aluminum, manganese, zinc, potassium, phosphorus, sulfur, etc.
Even after treatment with proteinase K, angelan has almost constant immune activity, showing that the immuno-stimulation principle of angelan resides in the polysaccharides alone or in combination with the inorganic compounds.
Quantitative and qualitative analyses for the sugars of angelan can be performed using thin layer chromatography (TLC) and ion exchange HPLC, respectively. The data demonstrate that the polysaccharides of angelan comprises galacturonic acid, galactose and arabinose as major components and mannose, rhamnose and xylose as minor components. That is, the composition of angelan, which consists mainly of galacturonic acid, arabinose and galactose, is typical of pectic polysaccharides which exist in a wide range of plants. Therefore, the major component of the immuno-stimulating principle of angelan is pectic polysaccharides and the immuno-stimulation of angelan is attributed to the pectic polysaccharides combined with the inorganic compounds. The immuno-stimulative polysaccharides isolated from Angelica gigas Nakai is 10 kDa or less in molecular weight.
Specific pathogen free mice for immune test were obtained from Korea Research Institute of Bioscience and Biotechnology, Korea. Investigation was done on the influence of angelan on the immune functions of the splenocytes including B cells, T cells and macrophages. Based on the data, the following conclusions were made.
The cellular targets in the angelan-induced immunopotentiation are divided into two types, i.e., direct and indirect targets. The direct exposure of angelan to splenocytes increases the expression of cytokines. The expression of IL-6, which is produced by activated macrophages, is enhanced by angelan. For IFN-xcex3, which is related to natural killer cells, a significant production increase can be induced by angelan. Also, IL-2 and IL-3, which are secreted from helper T cells, are expressed at an elevated level by angelan. However, the time-dependent analysis showed some differences in the onset time of angelan""s action among the four cytokines. IL-6 and IFN-xcex3 respond rapidly to angelan and the increased expression levels are maintained thereafter. In contrast, the production of IL-2 and IL-4 is weakly enhanced at delayed times. IL-2 production gradually increases and IL-4 production is affected for only a few hours after treatment with angelan. Accordingly, this suggests that macrophages and natural killer cells, which produce IL-6 and IFN-xcex3 respectively, are primary cellular targets directly affected by angelan whereas helper T cells are indirectly affected by angelan.
Angelan has an direct influence on B cells. Similar to LPS, which is a B cell mitogen, angelan causes a high level of mitogenic proliferation of B cells. When total splenocyte populations including B cells, T cell and macrophages are treated with angelan, they are proliferated at a significant increased level. In the absence of T cells and macrophages, angelan also augments the proliferation of B cells whereas the B cell-depleted populations are not affected, which suggests that B cells are directly activated by angelan. Mitogenicity comparison of angelan with specific mitogens, i.e., lipopolysaccharide (LPS) as a B cell mitogen, phytohemagglutinin (PHA) and concanavalin A (Con A) as T cell mitogens, and pokeweed mitogen (PWM) as a comitogen, demonstrates that angelan behaves like LPS. That is, angelan is a B cell mitogen.
The immuno-stimulative effects examined above also provide information to use angelan as an immuno-stimulating agent or drug. As described above, angelan enhances the antibody production of B cells. The antibody production of B cells are accomplished in two routes: polyclonal activation of B cells to produce IgM independent on other cell types and T-dependent antibody response of B cells requiring T cells, macrophages and helper T cells. Angelan induces increased antibody production in the two antibody responses and particularly, augments T-dependent antibody response from the early time of treatment. Therefore, angelan is thought to influence B cells and macrophages at once.
To examine the anticancer activity of angelan, mice are transplanted with cancer cells. A test group which is treated with angelan shows a higher viability than non-treated groups. This anticancer activity of angelan comes from its immuno-stimulating activity. Mouse""s immune functions stimulated by angelan defeat the cancer cells. Adriamicin, a typical anticancer chemical, is disadvantageous in that its high administration levels cause serious side effects with an insignificant therapeutic effect at low levels. According to the present invention, angelan can significantly reduce the administration dose of adriamicin with an improved anticancer effect, thereby preventing the side effects. In an in vivo experiment, co-administration of angelan and adriamicin at a dose of 0.3 mg/kg completely cured the mice affected with melanoma. In another experiment, angelan showed no cytotoxicity effect on cancer cells, implying that its anticancer effect comes from the enhancement in the immune defense rather than in cytotoxicity.
Using one or more pharmaceutically acceptable carriers, the angelan of the present invention may be formulated to pharmaceutical compositions in a routine manner. Suitable forms to be administrated via various routes, such as oral, rectal, subcutaneous, intravenous and intramuscular routes, include, for example, tablets, hard and soft gelatine capsules, liquor, emulsions, suspensions, suppositories, and ampules.
For the preparation of pharmaceutical compositions, angelan may be combined with therapeutically inactive inorganic or organic carriers. Lactose, corn starch or its derivatives, active stearic acid or its salts can be used as carriers for, e.g. capsules and hard gelatine capsules. Suitable carriers for soft gelatine capsules include, for example, vegetable oils, waxes, semi-solid and liquid polyols. If necessary, no carriers may be used. Examples of the carriers suitable to prepare liquor and syrups include water, alcohols, polyols, sucrose, invert sugars, and glucose. Examples of the carriers for ampules include water, polyols, glycerines and vegetable oils. For suppositories, natural and hardened oils, waxes, lipids, and semi-liquid polyols. In addition, the pharmaceutical composition of the present invention may contain preservatives, stabilizers, wetting agents, emulsifiers, sweetening agents, coloring agents, favors, osmosis-controlling salts, buffers, coating agents and/or antioxidants.
For the treatment of cancers, the angelan of the present invention may be administered at a wide range of dose. This administration dose may be adjusted according to personal conditions. In general, the pharmaceutically essential component of the composition may be daily administered at a dose of about 20 to 200 mg per kg of body weight for adults and preferably at about 30 mg. If necessary, the administration amount of angelan may exceed the upper limit. The daily dose may be administered at once or in installments unless the total amount is changed.